Overall: Project Summary/Abstract The hypothesis to be tested in our U19 Program Project is that combining therapies of gene-engineered cellular (chimeric antigen receptor (CAR)) and humoral (broadly neutralizing antibodies (bNAb)) immune reagents will lead to a cure of HIV-1 disease. In the single remarkable case of the ?Berlin patient?, allogeneic transplant of CCR5?32 donor cells resulted in a functional cure without evidence for remaining HIV-1. However, a universal unresolved limitation of transplant of gene-engineered hematopoietic stem/progenitor cells (HSPC) has been the difficulty of achieving engraftment levels sufficient to provide good therapeutic efficacy. We propose here to focus on building gene-engineered cellular and humoral immune therapeutics. One approach is a CAR recognizing HIV-1 infected cells. T cell immunotherapy with tumor specific CARs delivered by adoptive T cell therapy has proven to be effective against cancer in early human studies. We hypothesize that HSPC based delivery of CAR- T cells can enhance the number and functional responses of the resultant engineered T cells. To complement the engineering of T cell-mediated immunity, we also propose to engineer B cells to express bNAbs modified as single chain variants (scFv-Fc bNAb). BNAbs directed to HIV-1 have shown promise at suppressing viremia in animal models and clearing SHIV from the blood and tissues of infant rhesus macaques and human clinical studies show effective suppression. Nonetheless, bNAbs require multiple injections to maintain levels required to suppress virus in plasma and have not cleared virus in chronic infection. We will use novel lentiviral vectors gene-engineered to target delivery of scFv-Fc bNAb into B cells in vivo without ex vivo manipulation. We hypothesize that this strategy will promote bNAb production, biodistribution and activation/differentiation of gene- modified B cells to clear HIV-1 reservoirs. Finally, success of these gene-modifying therapeutics depends upon achieving sufficient systemic levels of gene-modified cells. Thus, another project will specifically address means to modulate up or down the levels of gene-modified cells to achieve maximum therapeutic efficacy. In addition, should any adverse effects be observed, the same reagent can be used to quickly eliminate gene-modified cells, providing a ?kill-switch?, and thus an added safety element to the overall approach. Drs. Kitchen and Chen (UCLA) will serve as dual-PIs. Both have extensive experience in general stem cell biology and its applications to HIV-1 disease. The Project/Core Leaders have expertise in HIV-1 biology and gene therapeutic approaches to HIV-1 disease. The breadth of expertise ranges from vector and transgene development (Chen, An, Morizono, Kitchen, Symonds), development and use of animal models for HSPC biology (Kitchen, An, Morizono, Chen, Kiem), anti-HIV-1 immune function (Yang, Kitchen), understanding of HSPC behavior (Chen) to clinical trial implementation (Symonds).